HER2Δ16 expression promotes estrogen independent and tamoxifen-resistant growth. (a–c) Graphs representing xenograft tumor kinetics of at least five nude mice per group injected with (a) MCF-7/Vector, (b) MCF-7/HER2 or (c) MCF-7/HER2Δ16 cells. With the exception of the E2 treatments, all mice were primed with E2 pellets and after 21 days, mice with established tumors were left untreated or implanted with TAM pellets. (d and e) MCF-7/HER2Δ16 cells are E2 independent and TAM resistant in vitro. (d and e) MCF-7/HER2Δ16 cells are estrogen independent and tamoxifen resistant in vitro. (d) Each cell line was untreated or treated for 5 days with 100 pM E2 alone or in combination with 1.0 μM TAM. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to quantitate cell growth. Results represent mean ± SE percent growth inhibition relative to 100 pM E2 alone. (e) Each cell line was cultured for 24 h in CS-MEM and treated for 72 h with 100 pM E2 alone or in combination with 1.0 μM TAM. Apoptosis was quantitated using a Cell Death Detection ELISA. Results represent apoptosis relative to cells treated with 100 pM E2 alone.

BCL-2 upregulation mediates tamoxifen resistance of MCF-7/HER2Δ16 cells. (a) Quantitation of estrogen-induced BCL-2 mRNA expression. Each cell line was treated the indicated time with 100 pM E2 alone or in combination with 1.0 μM TAM. Each RNA sample was analyzed in triplicate, normalized to a β-actin internal control and BCL-2 mRNA expression is represented relative to the untreated MCF-7/Vector. (b) BCL-2 protein is upregulated in tamoxifen-treated MCF-7/HER2Δ16 cells. Each cell line was cultured for 48 h in phenol red-free modified Eagle's medium containing 5% charcoal-stripped fetal bovine serum and then treated as above and cell lysates were analyzed by western blot for BCL-2 expression 24, 48 and 72 h after treatment. (c) Inactivation of ERα signaling induces BCL-2 protein upregulation in MCF-7/HER2Δ16 cells. The MCF-7/HER2Δ16 cell line was cultured for 48 h in and left untreated (-E2) or treated with 1.0 μM TAM or 100 nM ICI 18278 (ICI) for the indicated time. Cell lysates were prepared and analyzed by western blot for BCL-2 expression. In all cases, western blot analysis of α-tubulin was included as a loading control and images were quantitated using the Odyssey Infrared Imaging System software.

HER2Δ16 expression suppresses miR-15a and miR-16. (a) Expression of miR-15a and miR-16 is suppressed in MCF-7/HER2Δ16 cells. Total RNA was extracted and analyzed for miR-15a or miR-16 expression by qRT–PCR. Results from three independent RNA extractions are represented as mean ± SE expression relative to β-actin. The lower levels of miR-15a and miR-16 expression in the MCF-7/HER2Δ16 cells failed to reach significance (paired Student’s t-test; P = 0.07 and P = 0.08, respectively). (b) Expression of miR-15a and miR-16 is not altered by estrogen or tamoxifen. Each cell line was cultured for 48 h in phenol red-free modified Eagle;s medium containing 5% charcoal-stripped fetal bovine serum and then left untreated or treated for 16 h with 100 pM E2 alone or in combination with 1.0 μM TAM. Three independent total RNA extractions from each cell line were analyzed in triplicate for miR-15a and miR-16 expression by qRT–PCR. Results were normalized to β-actin and represented as mean ± SE expression relative to untreated MCF-7/Vector cells. Differences failed to obtain significance as determined by paired Student’s t-test.

Suppressed miR-15a/16 expression promotes tamoxifen resistance. (a) Inhibition of miR-15a/16 results in enhanced BCL-2 expression. Each cell line was treated with 50 nM of the indicated miR inhibitor for 48 h and BCL-2 expression was analyzed by western blot. Analysis of α-tubulin was included as a loading control and images were quantitated using the Odyssey Infrared Imaging System software. (b and c) Suppression of miR-15a/16 promotes tamoxifen resistance. Each cell line was untreated or treated with the indicated anti-miR and treated with 100 pM E2 alone or in combination with 1.0 μM TAM for five days. (b) 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to quantitate cell growth and (c) apoptosis was quantitated using a Cell Death Detection ELISA. Data are represented as mean ± SE of three independent experiments performed with triplicate samples relative to mock anti-miR and E2/TAM-treated MCF-7/Vector cells. Asterisks indicate samples with significant differences as determined by paired Student’s t-test (P < 0.008).